Advancesinscreeningtechnologieshavemadeliganddiscoveryagainstbiologicaltargetsroutine,but convertingbindingligandsintospecificenzymeinhibitorsisextremelychallenging,evenformetalloproteinases andotherenzymeswithwell-definedactivesites.Thelackofspecificinhibitorspreventsfullelucidationof biologicalprocessesasbasicasextracellularmatrixremodeling.Proteinsandsmallmoleculeseachlackkey featuresofinhibitors.Antibodiesandotherproteinsrarelydisruptenzymefunction,butusuallyexhibithigh bindingspecificity.Smallmoleculesfrequentlylacksingle-enzymespecificity,butinterferewithenzymatic activity.Neitherofthesemodalitiesiswell-suitedforgeneratingpotent,specificenzymeinhibitors. Mylong-termgoalsareto1)establishgeneralprinciplesfordiscoveringpotent,specificinhibitorsagainst medicallyrelevantenzymes;?and2)utilizetheresultinginhibitorstounderstandtherolesofenzymessuchas metalloproteinasesinnormalphysiologyandpathologicalprocesses.Ihypothesizethatsimultaneously leveragingthecomplementarystrengthsofproteinsandsmallmoleculeswillgiverisetoentirelynewclasses ofpotent,specificinhibitors.Thegoalduringthisproposalperiodistoconvertyeastdisplay,apowerful liganddiscoveryplatform,intoacomprehensiveinhibitordiscoveryplatform.Mylabhasalreadyestablished strategiesforexpandingthechemicalfunctionalitythatcanbeutilizedincombinationwithyeastdisplay.Here, wewillenhanceourplatformfurtheranduseittoidentifyinhibitorsagainstatestsetofmetalloproteinase targets.Intheprocess,wewillgainfundamentalinsightsintohowtogenerateinhibitorsthatarenotaccessible usinganycurrentinhibitordiscoveryapproaches,settingthestagefor1)agreatlyexpandedtoolkitforstudying basicbiology;?and2)muchbroaderinhibitordiscoveryefforts.Theinitialdirectionswewillpursueare: Direction1.Expandtherangeofchemistriesthatcanbeencodedinyeast-displayedproteins. Proteinscontainingcanonicalaminoacidslackkeygroupsfoundinenzymeinhibitors.Wewillutilizeour quantitativereporterofncAAincorporationtoencodethesefunctionalitiesinyeast-displayedproteins. Direction2.Establishassaysforquantitativelyevaluatingenzymeinhibitionontheyeastsurface. Noexistingdisplaytechnologiessupportquantitativeevaluationsofenzymeinhibitionduringhigh throughputscreening.Wewillutilizedualyeastdisplaytechnologytoestablishthesecapabilities. Direction3.Usechemicallyaugmentedantibodylibrariestoevolvepotent,specificinhibitors. Antibodiesrarelyinhibitenzymes.Wewillgenerateandscreenlibrariesofantibodiescontainingadded chemicalgroupstoestablishgeneralprinciplesforinhibitorisolationinthisunexploreddiscoveryspace. Tofocusourdiscoveryefforts,weandourcollaboratorshaveidentifiedmetalloproteinasesfrommultiple familiesthatplayimportantrolesinhumanhealthanddisease.Thegeneraldiscoveryprinciplesweestablish herewillleaddirectlytonewclassesofinhibitorsforunderstandingandtreatinghumandisease.